The present invention relates to a method for cryopreserving microencapsulated living animal cells enclosed in immunoisolation membranes, such microencapsulated living animal cells enclosed in the immunoisolation membranes, and biohybrid artificial organ modules using such microencapsulated living animal cells enclosed in the immunoisolation membranes. In particular, the present invention relates to a method for cryopreserving microencapsulated living hepatocytes in immunoisolation membranes, such microencapsulated living hepatocytes in the immunoisolation membranes, and biohybrid artificial organ modules using such microencapsulated living hepatocytes in the immunoisolation membranes.
Although acute hepatic failure has been still recognized as a serious disease among various ones, high survival rates have been recently reported for patients suffering high fulminant hepatic failure according to an artificial liver-assisting method based on a mechanically blood-purifying method in which plasmaphoresis and blood filtration/osmosis are combined. Thus, results on such treatments will be largely expected in the future. See (1) Yoshiba M, et al., “Favorable effect of new artificial liver support on survival of patients with fulminant hepatic failure.” Artif Organ. 20, pp. 1169-72, 1996. In Europe, hybrid type artificial liver modules have been clinically applied, aiming at further assistance of the liver function, and some of them succeeded as bridge-like or interface uses. See (2) Watanabe F D, Mullon C J, Hewitt W R, Arkadopoulos N, kahaku E, Eguchi S, Khalili T, Arnaout W. Shackleton C R, Rozga J, Solomon B, Demetriou A A; “Clinical experience with a bioartificial liver treatment of severe liver failure” and (3) Adam M., “Extracorporeal liver support: Waiting for the deciding vote.” ASAIO J. 49, 2003, pp. 621-632. In Japan, epoch-making artificial liver modules have been researched and developed. See (4) Mizumoto H, Funatsu K., “Liver regeneration using a hybrid artificial liver support system.” Artif Organs. 28, 2004, pp. 53-57 and (5) Kobayashi N, Okitsu T, Nakaji S, Tanaka N., “Hybrid bioartificial liver: establishing a reversibly immortalized human hepatocyte line and developing a bioartificial liver for practical use.” However, there may be problems in infection with swine retrovirus, overcoming of immunological reactions, adjustment of the number of hepatocytes in reactors, securement of cell sources, establishment of long-term cryopreserving methods, etc., and new artificial livers which can be clinically applied and exceed the existing treatment method for the acute hepatic failure have not been developed yet. See references (1) to (5) listed above.
It has been a common technical knowledge in the art that cells from living animal organs are slowly cooled, since rapid cooling resulting in damages of such cells must be avoided. The conventional recognition is that rapid freezing make a cell-suspended liquid form needle-shape crystals, so that thus tips of formed needle-shaped crystals pierce cells into death. Thus, according to some conventional techniques, hepatic cells are microencapsulated, and cryopreserved in a cryogenic freezer which can generally cool down to −80° C., while the cooling rate is being measured and controlled to 0.1 to 10° C./min, 0.1 to 100° C./min, or the like with a T thermocouple or the like with a voltage being controlled according to a program. Alternatively, based on the above conventional technical recognition, the cells are maintained at −70° C. for 24 hours after slow cooling in the cryogenic freezer, and then they are cooled with liquid nitrogen (freezing point: −196° C.). See references (6) to (9) below:
(6) Transplantation, 1993, Vol. 55, No. 3, pp. 616 to 622.
(7) Nippon Mechanics Academic Society Annual Conference Papers, 1999, Vol. 1999, No. 2, pp. 309-310.
(8) Nippon Mechanics Academic Society Papers B, 2001, Vol. 67, No. 654, pp. 580 to 587.
(9) Bioengineering Academic Conference Papers 1999, Vol. 11, pp. 96 to 97.
(10) Hepatology, 1989, Vol. 10, No. 5, pp. 855 to 860.
When hybrid type artificial organs such as hybrid type artificial livers and the like are considered, it is important to prevent immunological reactions. In addition, it is desirable that the function of the cells can be kept at a high level and a sufficient amount of such cells can be preserved for a long time. In order to solve the above problems, the present inventors tried to establish a method for cryopreserving living animal cells enclosed in immunoisolation membranes and then succeeded.